The present invention relates to solid phase synthetic methods, and more particularly, to solid support synthetic methods useful for producing combinatorial libraries of modulators of LXRs.
Cholesterol is used for the synthesis of bile acids in the liver, the manufacture and repair of cell membranes, and the synthesis of steroid hormones. There are both exogenous and endogenous sources of cholesterol. The average American consumes about 450 mg of cholesterol each day and produces an additional 500 to 1,000 mg in the liver and other tissues. Another source is the 500 to 1,000 mg of biliary cholesterol that is secreted into the intestine daily; about 50 percent is reabsorbed (enterohepatic circulation). Excess accumulation of cholesterol in the arterial walls can result in atherosclerosis that is characterized by plaque formation. The plaques inhibit blood flow and promote clot formation, and can ultimately cause heart attacks, stroke and claudication. Development of therapeutic agents for the treatment of atherosclerosis and other diseases associated with cholesterol metabolism has been focused on achieving a more complete understanding of the biochemical pathways involved. Most recently, liver X receptors (LXRs) were identified as key components in cholesterol homeostasis.
The LXRs were first identified as orphan members of the nuclear receptor superfamily whose ligands and functions were unknown. Two LXR proteins xcex1 and xcex2 are known to exist in mammals. The expression of LXRxcex1 is restricted, with the highest levels being found in the liver, and lower levels found in kidney, intestine, spleen, and adrenals. See, Willy, et al., Genes Dev. 9(9):1033-45 (1995). LXRxcex2 is rather ubiquitous, being found in nearly all tissues examined. Recent studies on the LXRs indicate that they are activated by certain naturally occurring, oxidized derivatives of cholesterol, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24,25(S)-epoxycholesterol. See, Lehmann, et al., J. Biol. Chem. 272(6):3137-3140 (1997). The expression pattern of LXRs and their oxysterol ligands provided the first hint that these receptors may play a role in cholesterol metabolism. See, Janowski, et al., Nature 383:728-731 (1996).
As noted above, cholesterol metabolism in mammals occurs via conversion into steroid hormones or bile acids. The role of LXRs in cholesterol homeostasis was first postulated to involve the pathway of bile acid synthesis, in which cholesterol 7xcex1-hydroxylase (CYP7xcex1) operates in a rate-limiting manner. Support for this proposal was provided when additional experiments found that the CYP7xcex1 promoter contained a functional LXR response element that could be activated by RXR/LXR heterodimers in an oxysterol- and retinoid-dependent manner.
Confirmation of LXR function as a transcriptional control point in cholesterol metabolism was made using knockout mice, particularly those lacking LXRxcex1. See, Peet, et al., Cell 93:693-704 (1998). Mice lacking the receptor LXRxcex1 (e.g., knockout or (xe2x88x92/xe2x88x92) mice) lost their ability to respond normally to increases in dietary cholesterol and were unable to tolerate any cholesterol in excess of that synthesized de novo. LXRxcex1 (xe2x88x92/xe2x88x92) mice did not induce transcription of the gene encoding CYP7xcex1 when fed diets containing additional cholesterol. This resulted in an accumulation of large amounts of cholesterol in the livers of LXRxcex1 (xe2x88x92/xe2x88x92) mice, and impaired hepatic function. These results further established the role of LXRxcex1 as the essential regulatory component of cholesterol homeostasis. LXRxcex1 is also believed to be involved in fatty acid synthesis. Accordingly, the discovery of new LXRxcex1 modulators such as antagonists, via screening methods could provide treatment for a variety of lipid disorders including obesity and diabetes.
High-throughput screening techniques allow for assaying the activity of thousands of molecules in short order. However, if molecules can only be synthesized one at a time, the rate of molecule submission to the assay becomes the rate-limiting step. To remedy this situation, various combinatorial techniques have been devised and implemented. Combinatorial chemistry is defined as the repetitive and systematic covalent attachment of different structural moieties to one another to produce a mixture of numerous distinct molecular entities or target molecules (i.e., combinatorial libraries). The desired target molecules include peptides, oligonucleotides, and small organic molecules. In general, combinatorial chemistry is utilized to generate a group of structurally related analogs that can then be evaluated to establish structure-activity relationships (SAR) and to optimize biological potency.
The importance of LXRs, and particularly LXRxcex1, to the delicate balance of cholesterol metabolism and fatty acid biosynthesis has led to the development of modulators of LXRs which are useful as therapeutic agents or diagnostic agents for the treatment of disorders associated with bile acid and cholesterol metabolism, including cholesterol gallstones, atherosclerosis, lipid storage diseases, obesity, and diabetes (see, co-pending application Ser. No. 09/479315, filed Jan. 6, 2000, incorporated herein by reference for all purposes). In view of the foregoing, there is a need in the art for combinatorial libraries of LXR modulators and the methods to produce them. The present invention fulfills this and other needs.
The importance of LXRs, and particularly LXRxcex1 to the delicate balance of cholesterol metabolism and fatty acid biosynthesis has led to the development of modulators of LXRs which are useful as therapeutic agents or diagnostic agents for the treatment of disorders associated with bile acid and cholesterol metabolism. However, more efficacious compounds are needed. As such, the present invention provides a method for preparing LXR ligands on a solid support, comprising:
(a) attaching an aniline derivative to the solid support to provide a support-bound aniline derivative;
(b) contacting the support-bound aniline derivative with an aldehyde or ketone under reductively aminating conditions to provide a support-bound substituted aniline derivative; and
(c) contacting the support-bound substituted aniline derivative with an acylating agent to provide an LXR ligand on the solid support. In certain preferred embodiments, the LXR ligand or modulator is cleaved or removed from the solid support.
The methods of the present invention enable the efficient generation of modulators of LXRs and other amide-derived products following cleavage from the support. In addition, the methods of the present invention can be used to generate diverse N-substituted benzanilide derivatives which, in turn, may be used in the formation of combinatorial libraries of compounds that can subsequently be screened for biological activity.
As such, the present invention provides a combinatorial library comprising compounds having the formula: 
wherein R1 is a group including, but not limited to, optionally substituted alkyl, optionally substituted aryl, optionally substituted (C8-C18)bicycloalkyl, optionally substituted (C8-C18)tricycloalkyl, optionally substituted (C8-C18)heterobicycloalkyl and optionally substituted (C8-C18)heterotricycloalkyl.
In a preferred embodiment, R1 is a functional group including, but not limited to, optionally substituted (C5-C18)cycloalkyl or a (C5-C18)heterocycloalkyl group, more preferably a (C8-C18)bicycloalkyl, (C8-C18)tricycloalkyl, (C8-C18)heterobicycloalkyl or (C8-C18)heterotricycloalkyl group. In particularly preferred embodiments, R1 represents an optionally substituted tricyclo[3.3.1.13,7]decanyl (or adamantyl), bicyclo[3.2.1]octanyl, bicyclo[5.2.0]nonanyl, bicyclo[4.3.2]undecanyl, tricyclo[2.2.1.01]heptanyl, tricyclo[5.3.1.11]dodecanyl, tricyclo[5.4.0.02,9]undecanyl, tricyclo[5.3.2.04,9]dodecanyl, tricyclo[4.4.1.11,5]dodecanyl or tricyclo[5.5.1.03,11]tridecanyl group. More preferably, R1 is a substituted or unsubstituted adamantyl group, most preferably an unsubstituted 1-adamantyl group.
R2 is a group including, but not limited to, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl and optionally substituted heteroarylalkyl. Preferred embodiments are those in which R2 is aryl(C1-C8)alkyl or heteroaryl(C1-C8)alkyl. More preferably, R2 is branched heteroaryl(C2-C8)alkyl, for example, 1-(furan-2-yl)ethyl, 1-(pyridin-2-yl)ethyl, 1-(furan-2-yl)-2-propyl, 1-(2-pyridyl)-2-propyl, 1-(furanyl)isobutyl, 1-(3-pyridyl)isobutyl, 1-(pyridin-4-yl)ethyl, 1-(pyridin-4-yl)isobutyl, and the like. Most preferably, R2 is 1-(furan-2-yl)ethyl or 1-(pyridin-2-yl)ethyl. In still other preferred embodiments, R2 is a branched (C3-C8)alkyl, more preferably an isopropyl group. In yet other preferred embodiments, R2 is a heteroaryl(C3-C8)alkenyl group. More preferably, R2 is a 1-(3-furanyl)-3-butenyl group.
X is a functional group including, but not limited to, xe2x80x94CO2R11, xe2x80x94CH2OR11, xe2x80x94C(O)R11, xe2x80x94C(O)NR11R12 and xe2x80x94CH2NR11R12, wherein R11 and R12 are each members independently selected from hydrogen and optionally substituted (C1-C8)alkyl. In certain aspects, the present invention also provides a combinatorial library that contains substituted benzanilides, wherein the benzanilides are optionally connected to a solid support.
In yet another aspect, the present invention provides methods for synthesizing libraries of substituted benzanilides having the formula: 
wherein
R1 is a group including, but not limited to, optionally substituted (C5-C18)cycloalkyl or a (C5-C18)heterocycloalkyl group, more preferably a (C8-C18)bicycloalkyl, (C8-C18)tricycloalkyl, (C8-C18)heterobicycloalkyl or (C8-C18)heterotricycloalkyl group. In particularly preferred embodiments, R1 represents an optionally substituted tricyclo[3.3.1.13,7]decanyl (or adamantyl), bicyclo[3.2.1]octanyl, bicyclo[5.2.0]nonanyl, bicyclo[4.3.2]undecanyl, tricyclo[2.2.1.01]heptanyl, tricyclo [5.3.1.11,5]dodecanyl, tricyclo[5.4.0.02,9]undecanyl, tricyclo[5.3.2.04,9]dodecanyl, tricyclo[4.4.1.11,5]dodecanyl or tricyclo[5.5.1.03,11]tridecanyl group. More preferably, R1 is a substituted or unsubstituted adamantyl group, most preferably an unsubstituted 1-adamantyl group.
R2 is a group including, but not limited to, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl and optionally substituted heteroarylalkyl. Preferred embodiments are those in which R2 is aryl(C1-C8)alkyl or heteroaryl(C1-C8)alkyl. More preferably, R2 is branched heteroaryl(C2-C8)alkyl, for example, 1-(furan-2-yl)ethyl, 1-(pyridin-2-yl)ethyl, 1-(furan-2-yl)-2-propyl, 1-(2-pyridyl)-2-propyl, 1-(furanyl)isobutyl, 1-(3-pyridyl)isobutyl, 1-(pyridin-4-yl)ethyl, 1-(pyridin-4-yl)isobutyl, and the like. Most preferably, R2 is 1-(furan-2-yl)ethyl or 1-(pyridin-2-yl)ethyl. In still other preferred embodiments, R2 is a branched (C3-C8)alkyl, more preferably an isopropyl group. In yet other preferred embodiments, R2 is a heteroaryl(C3-C8)alkenyl group. More preferably, R2 is a 1-(3-furanyl)-3-butenyl group.
X is a group including, but not limited to, xe2x80x94CO2R11, xe2x80x94CH2OR11, xe2x80x94C(O)R11, xe2x80x94C(O)NR11R12 and xe2x80x94CH2NR11R12, wherein R11 and R12 are each members independently selected from hydrogen and optionally substituted (C1-C8)alkyl. In certain aspects, the present invention also provides a combinatorial library that contains substituted benzanilides, wherein the benzanilides are optionally connected to a solid support.
The present invention also provides a method for screening a library that contains a substituted benzanilide of Formula I optionally connected to a solid support.
These and other features and advantages will become more apparent when read with the accompanying drawings and detailed description that follow.